The tumor suppressor PTEN plays important roles in the control of apoptosis, cell cycles and development by acting in conjuction with PI3-kinase to regulate the levels of PI 3,4,5-P3. However, how the activity of PTEN is regulated in response to extracellular stimuli is not known. Overexpression of PTEN prevents basal level activation of Akt, a PI 3,4,5-P3 effector, in the absence of growth factor stimulation. However, growth factor stimulation of these cells results in activation of Akt via a PI3-Kinase-dependent pathway. This suggest that there are mechanisms by which PTEN activity is suppressed and PIP3 accumulates in cells stimulated with a growth factor. Increasing evidence suggests that superoxide and H2O2 are transiently produced in response to various extracellular stimuli and their intracellular levels are controlled by the generating enzymes like NADPH oxidases and lipooxygenases as well as the scavenging enzymes like superoxide dismutases, catalases, and peroxiredoxins (Prxs).PTEN has an essential Cys124 at the active site. Incubation with H2O2 resulted in the inactivation of purified PTEN in a manner dependent on time and H2O2 concentration. Studies of various Cys mutants and direct mass spectral analysis of tryptic peptides indicated that Cys124 formed a disulfide with Cys71 upon oxidation with H2O2 and superoxide anion. The H2O2-inactivated PTEN was incubated with various electron donors [DTT, GSH, thioredoxin (Trx), glutaredoxin (Grx)] and reactivation was monitored. The most rapid reactivation was achieved by DTT and Trx (5 microM), whereas GSH (5 mM) was the least efficient. The sensitivity of PTEN to receptor-produced H2O2 was tested in human neutrophils stimulated with fMLP. The amount of oxidized PTEN increased with time of incubation with fMLP, reaching a maximum at 5 min, and returned to the basal value by 20 min, suggesting that the PTEN oxidation is reversible. Incubation of cells with 1-chloro-2,4-dinitrobenzene, an inhibitor of thioredoxin reductase caused the conversion of reduced PTEN to oxidized form, whereas buthioninesulfoximine, an inhibitor of glutathione biosynthesis did not have any effect. These results suggest that the activity of PTEN is regulated through reversible oxidation by H2O2 and the reduction is achieved mainly by thioredoxin.